EP0616035A2 - Transgenic pathogen resistant organism - Google Patents

Transgenic pathogen resistant organism Download PDF

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Publication number
EP0616035A2
EP0616035A2 EP93116011A EP93116011A EP0616035A2 EP 0616035 A2 EP0616035 A2 EP 0616035A2 EP 93116011 A EP93116011 A EP 93116011A EP 93116011 A EP93116011 A EP 93116011A EP 0616035 A2 EP0616035 A2 EP 0616035A2
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Prior art keywords
pathogen
organism
transgenic
gene
resistant
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German (de)
French (fr)
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EP0616035B1 (en
EP0616035A3 (en
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Jürgen Dr. Logemann
Guido Jach
Birgit Görnhardt
John Dr. Mundy
Jeff Prof. Schell
Peter Dr. Eckes
Ilan Prof. Chet
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S47/00—Plant husbandry
    • Y10S47/01—Methods of plant-breeding and including chromosome multiplication

Definitions

  • the invention relates to a pathogen-resistant organism and a method for its production.
  • pathogens infest a plant with a number of different reactions. These include, for example, changes in the cell wall structure, the synthesis of antimicrobial phytoalexins, the accumulation of so-called PR proteins ("pathogenesis-related"), protease inhibitors and enzymes with hydrolytic functions (Hahlbrock and Grisebach in Ann. Rev. Plant. Physiol ., 30 , (1979), 105-130).
  • PR proteins pathogenesis-related
  • the invention is based on the surprising finding that the incorporation into the genome of an organism of at least two different genes with a pathogen-inhibiting effect helps the organism to become resistant to pathogens, which goes far beyond an additive effect of the genes in each case.
  • the genes can code for gene products that reduce the vitality of fungi.
  • the genes can be of fungal, bacterial and plant, animal or viral origin.
  • the gene products have properties that promote fungal resistance.
  • the gene products are chitinase (ChiS, ChiG), glucanase (GluG), protein synthesis inhibitor (PSI) and antifungal protein (AFP).
  • the transgenic pathogen-resistant organism can be a plant, preferably tobacco, potato, strawberry, corn, rapeseed or tomato plants.
  • the invention also relates to DNA transmission vectors with inserted DNA sequences, as detailed in this description.
  • a method for the production of pathogen-resistant organisms is the subject of the invention, wherein vectors are used for the transformation into the genome of an organism which comprise more than one gene with pathogen-inhibiting effect.
  • the invention also relates to a method for ensuring the resistance of organisms to pathogens, characterized in that the organism is a transgenic pathogen-resistant organism according to one of Claims 1 to 7 or an organism whose genome comprises at least one gene according to those used here Contains definitions (see claims 1 to 7), used and applied to the organism at least one substance which is not expressed by the organism but which corresponds to any other of the gene products according to the definitions given in this application (claims 1 to 7).
  • the PSI gene comes from barley and codes for a protein that inhibits the protein synthesis of fungi. According to in vitro tests, low concentrations of PSI are sufficient to inhibit various fungi such as Rhizoctonia solani.
  • a polypeptide can be isolated and sequenced from the fermentation broth of Aspergillus giganteus, which via antifungal Activity.
  • This polypeptide is suitable as an antifungal agent, for example as a spray and as a preservative for technical products and food and feed. It can also be combined with other pesticidally active substances, fertilizers or growth regulators. Inhibitory activities against fungi could be detected against various Aspergillus, Fusarien, Phytophthora and Trichophyton species.
  • ChiG chitinase
  • GluG glucanase
  • Purified ChiG protein or GluG protein inhibits various phytopathogenic fungi in vitro (including Rhizoctonia solani) (see R. Leah et al., Journal of Biological Chemistry, Vol. 266, No. 3 (1991), pages 1564-1573).
  • the inventors have now found, completely surprisingly, that the at least two-fold combined expression of PSI, AFP, ChiS, ChiG or GluG leads to synergistic effects in transgenic plants with regard to the acquired fungal resistance.
  • the effects of the individual substances in the combination are clearly exceeded. These include resistance to the fungus Rhizoctonia solani, Sclerotinia infestation, Botrytis infestation, etc.
  • Combinations according to the invention are (DNA and / or polypeptides):
  • the invention further relates to the combined use of the proteins with pathogen-inhibiting activity, preferably ChiS, PSI, AFP, ChiG and GluG, against pathogens.
  • Combined use also means that at least one first pathogenic inhibiting substance is expressed by the organism and at least one second substance which has a pathogenic inhibiting effect is applied to the organism from the outside.
  • the agents according to the invention also include those which contain the abovementioned proteins in at least two combinations.
  • the agents according to the invention can contain further active ingredients.
  • These further active ingredients can be pesticides, fertilizers and / or growth regulators, the agents according to the invention can also be provided in different formulations, such as concentrates, emulsions, powders, formulations on carriers, mixtures with other active ingredients, etc.
  • the combination ChiS / PSI is particularly preferred and AFP / PSI.
  • These proteins can be used particularly effectively to inhibit the growth of Rhizoctonia solani, in particular in tobacco plant crops.
  • the invention also relates to the use of a DNA sequence in a method according to the invention which codes at least for a polypeptide of the sequences A to E, or a pathogen-resistant organism, the genome of which has at least two different genes under the control of active promoters with pathogen contains an inhibitory effect, the genes each being selected from the group of sequences A to E.
  • the invention further includes DNA sequences which hybridize with a DNA sequence which codes for polypeptides of the amino acid sequences A to E, which DNA sequences can be of natural synthetic or semi-synthetic origin and with the aforementioned DNA sequence by mutations, Nucleotide substitutions, nucleotide deletions, Nucleotide insertions and inversions of nucleotide sequences can be related and encoded for a polypeptide with pathogenic activity.
  • the invention furthermore relates to a recombinant DNA molecule which contains at least one DNA sequence as described above, it being possible for this DNA molecule to be present as a cloning or expression vector.
  • the invention relates to corresponding host organisms and intermediate hosts which have been transformed with a recombinant DNA molecule as described above.
  • Bakery strains are preferred as intermediate hosts in the production of a pathogen-resistant transgenic organism, in particular so-called agrobacterial strains.
  • the invention further relates to the transgenic pathogen-resistant organisms obtained by the process according to the invention, in particular tobacco, potato, corn, pea, rape and tomato plants.
  • the DNA sequences according to the invention are generally transferred together with a promoter.
  • Promoter sequences are recognized by the plant transcription apparatus and thus lead to a constitutive expression of the gene associated with them in plants.
  • the promoter can also be pathogen-inducible and / or wound-inducible (WUN1) and / or tissue-specific and / or development-specific.
  • the DNA coding for the amino acid sequences A to E was first cloned in a manner known per se and then transferred by conjugation to A. Tumefaciens LBA 4404 (A. Hoekema et al., Nature 303 , 179-180). This was done according to the Van Haute et al. method described in EMBO J. 2 , 411-418 (1983).
  • the DNA transfers into the agrobacterium were checked by isolating agrobacterial DNA according to the method described by Ebert et al. in proc. Natl. Acad. Sci. USA 84 5745-5749 (1987), method described.
  • the restriction cleavage of the DNA, the transfer to Hybond-N membrane (Amersham) and the hybridization against a radioactively labeled DNA probe provided information about a successful DNA transfer into the agrobacterium.
  • Tobacco, rapeseed, strawberry, tomato and potato plants were in turn transformed using the transformed agrobacterium.
  • MSC16 MS + 0.5 ⁇ g / ml BAP + 0.1 ⁇ g / ml NAA + 100 ⁇ g / ml kanamycin sulfate + 500 ⁇ g / ml Claforan) transferred.
  • MSC15 MS + 2% sucrose, 500 ⁇ g / ml claforan + 100 ⁇ g / ml kanamycin sulfate). Root-shaped shoots were further analyzed.
  • Monocotyledonous plants including maize
  • some diclyte plants were transformed into protoplasts using direct gene transfer. These protoplasts were then regenerated to intact plants (example: J. Potrykus in Biotechnology 8 (1990) 535).
  • the transgenic plants obtained were infected with the fungus Rhizoctonia solani for test purposes.
  • mushroom cultures were grown and mixed thoroughly in common soil. This soil was then distributed in a bowl and planted with the plants to be tested.
  • Rhizoctonia solani mushroom mycelium was mixed with 100 ⁇ l potato dextrose solution and incubated in microtiter plates at 25 ° C.
  • the growth of the fungus correlates linearly with the increase in optical density at 405 nanometers.
  • the inhibitory effect of proteins can be demonstrated by a smaller increase in optical density.
  • E W1 + W2 - ((W1 X W2) / 100)
  • W1 and W2 indicate the efficiencies of the individual proteins, which means the percentage deviation of the growth curve (in the presence of the protein) from the untreated control.
  • OD (K) is the optical density of the untreated control
  • OD (P) is the optical density of the culture treated with the protein.
  • the proteins ChiS, PSI, AFP, ChiG and GluG used in the example surprisingly showed synergistic inhibitory effects against various fungi, these effects being achieved both by the combination of two types of protein and by the multiple combination of the above-mentioned proteins.
  • Fig. 1 shows the results obtained with the combination of the proteins as well as with the individual substances. According to the figure, different ChiS concentrations (0.5 ⁇ g / ml or 0.05 ⁇ g / ml) are combined with PSi protein (1.0 ⁇ g / ml).
  • transgenic plants were first generated which contained at least one of the genes interacting synergistically.
  • a ChiS gene was first fused with plant regulatory sequences.
  • a 1.8 Kb ChiS gene was generated using synthetic oligonucleotides according to the dideoxy sequencing method of Sanger et al. in proc. Natl. Acad. Sci. USA, 74 , (1977), 5463-5467.
  • the 35S promoter 400 bp (according to Töpfer et al. In Nucl. Acid. Res., 15 (1987) 5890)) derived from the cauliflower mosaic virus (CamV) was transcriptionally fused with the ChiS gene. 3 'from the ChiS gene the 0.2 Kb termination signal of the 35S gene of the CamV was used, the functionality of which is known in dicotyledonous plants.
  • the chimeric gene 35S-ChiS was cloned into the vector pLS034, transformed into tobacco and potato plants by means of the Agrobacterium tumefaciens transformation system, and kanamycin-resistant plants were regenerated.
  • PolyA+ ⁇ RNA was first isolated from ripe barley seeds (Hordeum vulgare L. cv. Piggy) and stored in a cDNA library in ⁇ -gt-11 phages. The details of the procedure are R. Lea in Plant. Biol., 12 (1989), 673-682. Monospecific PSI antibodies were then used to identify cDNA clones.
  • the transferred gene as well as mRNA and gene product could be detected in corresponding transgenic tobacco, potato, rapeseed, strawberry and tomato plants.
  • the cDNA sequence of the antifungal peptide is terminated for cloning in the vector which can be ligated into BamH1 and Sal1 restriction sites.
  • PDH51 (Pietrzak et al. In Nucl. Acids Res. 14 (1986), 5857) was used as the cloning vector.
  • the vector pDH51 was opened with the restriction enzymes BamH1 and Sal1 between the promoter and terminator.
  • the vector pDH51 is a pUC18 derivative, the promoter and terminator sequences of the 35S transcript Contains cauliflower mosaic virus. These sequences are recognized by the plant transcription apparatus and lead to a strong constitutive expression of the gene associated with them in plants.
  • the DNA of the antifungal peptide is then cloned into the vector via the BamH1 and Sal1 cleavage site. Finally, the transcription unit - promoter, gene and terminator - is cut out of the vector with the restriction enzyme EcoRI and cloned into a plant transformation vector.
  • the following vectors or their derivatives, for example, can be used as the plant transformation vector: pOCA18 (Olszewski et al. in Nucl. Acids Res., 16 (1988), 10765) pPCV310 (Koncz and Shell in MGG 204 (1986), 383) and pBin19 (Bevan et al. Nucl. Acids. Res.
  • Transgenic tobacco, potato, strawberry, rapeseed and tomato plants were then transformed using the method described above. Transformed shoots are selected for their resistance to the antibiotic kanamycin.
  • the expression of the antifungal protein in the transformed useful plants was checked and confirmed by DNA analysis (Southern blotting), RNA analysis (Northern blotting) and protein analysis with specific antibodies (Western blotting).
  • ChiG or GluG transgenic plants were obtained which were both Southern, Northern and Western positive.
  • the aforementioned genes could be integrated into the genome of monocotyledonous plants such as maize by means of direct gene transfer. In this way, transgenic plants were obtained which were both Southern, Northern and Western positive.
  • a tobacco plant that expresses ChiS and PSI shows a much greater resistance to Rhizoctonia attack than the plants that either only expressed ChiS or PSI or that would result from the additive resistance.
  • a synergistic inhibitory effect also results from the combined expression of PSI and AFP transgenic tobacco against Rhizoctonia solani infestation.
  • the two or more combinations of different genes (ChiS, RIP, AFP, ChiG and GluG) in a wide variety of transgenic plants also led to synergistic inhibitory effects against various fungi.

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Abstract

New transgenic, pathogen-resistant organisms have a genome contg. at least 2 different genes, under control of active promoters, with pathogen-inhibiting activity. Also new are DNA transfer vectors contg. inserted DNA sequences for prepn. of such organisms. Partic. the genes (of fungal, bacterial, animal, plant or viral origin) encode products that reduce the viability of fungi, esp. chitinase; glucanase; protein-synthesis inhibitor (PSI) or antifungal protein (AFP). The transgenic organism is pref. a plant, esp. tobacco, potato, strawberry, maize, rape or tomato. The specification includes nucleic acid and amino acid sequences for AFP of Aspergilllus giganteus (275 bp); PSI of Hordeum vulgare (1079 bp); exo-chitinase (ChiS) of Serratia marcescens (2329 bp); chitinase G of H. vulgare (1013 bp), and glucanase of H. vulgare (1249 bp).

Description

Die Erfindung betrifft einen pathogen-resistenten Organismus sowie ein Verfahren zu dessen Erzeugung.The invention relates to a pathogen-resistant organism and a method for its production.

Im Stand der Technik ist bekannt, daß der Befall einer Pflanze durch Pathogene eine Reihe verschiedener Reaktionen hervorgerufen werden. Dazu gehören zum Beispiel Veränderungen in der Zellwandstruktur, die Synthese antimikrobiell wirkender Phytoalexine, die Akkumulation von sogenannten PR-Proteinen ("Pathogenesis-related"), Protease-Inhibitoren und Enzyme mit hydrolytischen Funktionen (Hahlbrock und Grisebach in Ann. Rev. Plant. Physiol., 30, (1979), 105-130).It is known in the prior art that pathogens infest a plant with a number of different reactions. These include, for example, changes in the cell wall structure, the synthesis of antimicrobial phytoalexins, the accumulation of so-called PR proteins ("pathogenesis-related"), protease inhibitors and enzymes with hydrolytic functions (Hahlbrock and Grisebach in Ann. Rev. Plant. Physiol ., 30 , (1979), 105-130).

Viele Pathogene (Pilze und Insekten) weisen als Bestandteil ihrer Zellwand Chitin auf. Demgegenüber besitzen Plfanzen kein Chitin. Es ist nun in einigen Fällen nachgewiesen worden, daß Plfanzen nach einem pathogenen Befall verstärkt Chitinasen produzieren. Chitinasen gehören zu den Enzymen mit hydrolytischen Funktionen und katalysieren den Chitinabbau. Es konnte nun gezeigt werden, daß Pflanzen durch die Produktion von Chitinasen eine erhöhte Widerstandsfähigkeit gegen Pathogene erhalten.Many pathogens (fungi and insects) have chitin as part of their cell wall. In contrast, plants have no chitin. It has now been shown in some cases that plants produce increased chitinases after a pathogenic attack. Chitinases are enzymes with hydrolytic functions and catalyze the breakdown of chitin. It has now been shown that the production of chitinases gives plants increased resistance to pathogens.

Weiterhin ist die Verwendung eines Gens aus Gerstenpflanzen bekannt, dessen Genprodukt für einen Inhibitor der pilzlichen Proteinsynthese kodiert. Der Einbau eines entsprechenden Inhibitorgens in transgenen Pflanzen führte zu einer verbesserten Pilzresistenz.Furthermore, the use of a gene from barley plants is known, the gene product of which codes for an inhibitor of fungal protein synthesis. The incorporation of a corresponding inhibitor gene in transgenic plants led to an improved fungal resistance.

Schließlich ist auch bekannt geworden, daß die Verwendung eines Polypeptids aus Aspergillus giganteus aufgrund dessen antifungaler Aktivität Pflanzen vor einem Pilzbefall schützen kann.Finally, it has also become known that the use of a polypeptide from Aspergillus giganteus, because of its antifungal activity, can protect plants from fungal attack.

Gegenüber diesem Stand der Technik besteht aber das Bedürfnis nach der Schaffung weiterer transgener pathogen-resistenter Organismen. Daneben sind solche Organismen besonders erwünscht, deren Resistenz gegenüber den bekannten Organismen insgesamt vergrößert oder bezüglich der Anzahl der möglichen Pathogene verbreitert wird.Compared to this prior art, however, there is a need for the creation of further transgenic pathogen-resistant organisms. In addition, those organisms are particularly desirable whose resistance to the known organisms is increased overall or the number of possible pathogens is broadened.

Dieses Problem wird durch einen transgenen pathogen-resistenten Organismus mit den Merkmalen des Anspruchs 1 gelöst.This problem is solved by a transgenic pathogen-resistant organism with the features of claim 1.

Der Erfindung liegt der überraschende Befund zugrunde, daß der Einbau mindestens zweier unterschiedlicher Gene mit pathogen-inhibierender Wirkung in das Genom eines Organismus diesem zu einer Resistenz gegen Pathogene verhilft, die weit über eine additive Wirkung der Gene jeweils für sich hinausgeht.The invention is based on the surprising finding that the incorporation into the genome of an organism of at least two different genes with a pathogen-inhibiting effect helps the organism to become resistant to pathogens, which goes far beyond an additive effect of the genes in each case.

In den Unteransprüchen werden weitere Ausführungsformen der Erfindung angegeben.Further embodiments of the invention are specified in the subclaims.

Die Gene können für Genprodukte kodieren, die die Vitalität von Pilzen herabsetzen. Insbesondere können die Gene pilzlichen, bakteriellen und pflanzlichen, tierischen oder viralen Ursprungs sein. Insbesondere haben die Genprodukte die Pilzresistenz fördernde Eigenschaften. Die Genprodukte sind Chitinase (ChiS, ChiG), Glukanase (GluG), Proteinsynthese-Inhibitor (PSI) und antifungales Protein (AFP).The genes can code for gene products that reduce the vitality of fungi. In particular, the genes can be of fungal, bacterial and plant, animal or viral origin. In particular, the gene products have properties that promote fungal resistance. The gene products are chitinase (ChiS, ChiG), glucanase (GluG), protein synthesis inhibitor (PSI) and antifungal protein (AFP).

Der transgene pathogen-resistente Organismus kann eine Pflanze sein, vorzugsweise handelt es sich um Tabak-, Kartoffel-, Erdbeer-, Mais-, Raps- oder Tomatenpflanzen.The transgenic pathogen-resistant organism can be a plant, preferably tobacco, potato, strawberry, corn, rapeseed or tomato plants.

Gegenstand der Erfindung sind auch DNA-Übertragungsvektoren mit inserierten DNA-Sequenzen, wie sie in dieser Beschreibung im einzelnen angegeben sind.The invention also relates to DNA transmission vectors with inserted DNA sequences, as detailed in this description.

Gegenstand der Erfindung ist weiterhin ein Verfahren zur Erzeugung pathogen-resistenter Organismen, wie sie hier beschrieben werden, wobei in das Genom eines Organismus mindestens 1 Gen mit pathogen-inhibierender Wirkung transferiert und der pathogen-resistente Organismus

  • (a) durch Kreuzung des Organismus mit einem gegebenenfalls transgenen weiteren Organismus, der mindestens ein anderes Gen mit pathogen-inhibierender Wirkung enthält, und anschließende Selektion und/oder
  • (b) durch Transformation dieses anderen Gens mit pathogen-inhibierender Wirkung in den Organismus erhalten wird. Das Verfahren kann mit DNA-Übertragungsvektoren mit inserierten DNA-Sequenzen entsprechend einem Gen mit pathogen-inhibierender Wirkung, wie hier beschrieben, verwendet werden.
The invention further relates to a method for producing pathogen-resistant organisms, as described here, wherein at least 1 gene with pathogen-inhibiting activity is transferred into the genome of an organism and the pathogen-resistant organism
  • (a) by crossing the organism with an optionally transgenic further organism which contains at least one other gene with a pathogen-inhibiting effect, and subsequent selection and / or
  • (b) is obtained by transforming this other gene with pathogen inhibiting activity into the organism. The method can be used with DNA transfer vectors with inserted DNA sequences corresponding to a gene with a pathogen-inhibiting effect, as described here.

Schließlich ist ein Verfahren zur Erzeugung pathogen-resistenter Organismen Gegenstand der Erfindung, wobei zur Transformation in das Genom eines Organismus Vektoren verwendet werden, die mehr als ein Gen mit pathogen-inhibierender Wirkung umfassen.Finally, a method for the production of pathogen-resistant organisms is the subject of the invention, wherein vectors are used for the transformation into the genome of an organism which comprise more than one gene with pathogen-inhibiting effect.

Gegenstand der Erfindung ist auch ein Verfahren zur Gewährleistung der Resistenz von Organismen gegen Pathogene, dadurch gekennzeichnet, daß als Organismus ein transgener pathogen-resistenter Organismus nach einem der Ansprüche 1 bis 7 oder ein Organismus, dessen Genom mindestens ein Gen gemäß den hier verwendeten Definitionen (siehe Ansprüche 1 bis 7) enthält, verwendet und auf den Organismus mindestens eine Substanz aufgebracht wird, die nicht durch den Organismus exprimiert wird, aber irgendeinem anderen der Genprodukte gemäß den in dieser Anmeldung gegebenen Definitionen (Ansprüche 1 bis 7) entspricht.The invention also relates to a method for ensuring the resistance of organisms to pathogens, characterized in that the organism is a transgenic pathogen-resistant organism according to one of Claims 1 to 7 or an organism whose genome comprises at least one gene according to those used here Contains definitions (see claims 1 to 7), used and applied to the organism at least one substance which is not expressed by the organism but which corresponds to any other of the gene products according to the definitions given in this application (claims 1 to 7).

Die synergistischen Wirkungen konnten ganz besonders mit transgenen pathogen-resistenten Organismen erzielt werden, auf die Gensequenzen transferiert oder transfiziert waren, die für Proteine der anhängenden Sequenzprotokolle A bis E kodierten bzw. diesen entsprachen.The synergistic effects could be achieved particularly with transgenic pathogen-resistant organisms to which gene sequences were transferred or transfected, which encoded or corresponded to proteins of the attached sequence listing A to E.

ChiS:ChiS:

Aus dem Bodenbakterium Serratia marcescens wurde ein 1,8 Kb großes DNA Fragment isoliert, daß für eine Chitinase, genannt ChiS, kodiert. In vitro Untersuchungen mit gereinigtem ChiS-Protein zeigten, daß es in geringen Konzentrationen bereits das Wachstum von Pilzen wirksam inhibieren kann. Die Ursache für die Inhibition ist, daß das ChiS-Protein über eine Chitinaseaktivität verfügt, mit der die Hyphenspitzen des Pilzes zerstört werden können. Auf diese Weise kann der Pilz nicht weiterwachsen und wird inhibiert.A 1.8 Kb DNA fragment was isolated from the soil bacterium Serratia marcescens, which codes for a chitinase, called ChiS. In vitro studies with purified ChiS protein showed that it can effectively inhibit the growth of fungi even in low concentrations. The reason for the inhibition is that the ChiS protein has a chitinase activity with which the hyphens of the fungus can be destroyed. In this way the fungus cannot continue to grow and is inhibited.

PSI:PSI:

Das PSI-Gen stammt aus Gerste und kodiert für ein Protein, welches die Proteinsynthese von Pilzen inhibiert. Nach in vitro Tests reichen bereits geringe Konzentrationen PSI aus, um diverse Pilze wie zum Beispiel Rhizoctonia solani zu inhibieren.The PSI gene comes from barley and codes for a protein that inhibits the protein synthesis of fungi. According to in vitro tests, low concentrations of PSI are sufficient to inhibit various fungi such as Rhizoctonia solani.

AFP:AFP:

Aus der Fermentationsbrühe von Aspergillus giganteus kann ein Polypeptid isoliert und sequenziert werden, welches über antifungale Aktivität verfügt. Dieses Polypeptid eignet sich als antifungales Agens, z.B. als Sprühmittel und als Konservierungsstoff für technische Produkte und Nahrungs- und Futtermittel. Es kann weiterhin mit anderen pestizid wirksamen Stoffen, Düngemitteln oder Wachstumsregulatoren kombiniert werden. Inhibitorische Aktivitäten gegen Pilze konnten unter Anderem gegen verschiedene Aspergillus-, Fusarien-, Phytophthora- und Trichophyton-Arten nachgewiesen werden.A polypeptide can be isolated and sequenced from the fermentation broth of Aspergillus giganteus, which via antifungal Activity. This polypeptide is suitable as an antifungal agent, for example as a spray and as a preservative for technical products and food and feed. It can also be combined with other pesticidally active substances, fertilizers or growth regulators. Inhibitory activities against fungi could be detected against various Aspergillus, Fusarien, Phytophthora and Trichophyton species.

ChiG und GluG:ChiG and GluG:

Aus bestimmten Gerstearten lassen sich zwei Gene isolieren, die für eine Chitinase (ChiG) bzw. Glukanase (GluG) kodieren. Gereinigtes ChiG-Protein oder GluG-Protein inhibiert in vitro diverse pflanzenpathogene Pilze (u.A. Rhizoctonia solani) (siehe R. Leah et al., Journal of Biological Chemistry, Vol. 266, No. 3 (1991), Seiten 1564-1573).Two genes can be isolated from certain types of barley, which code for a chitinase (ChiG) or glucanase (GluG). Purified ChiG protein or GluG protein inhibits various phytopathogenic fungi in vitro (including Rhizoctonia solani) (see R. Leah et al., Journal of Biological Chemistry, Vol. 266, No. 3 (1991), pages 1564-1573).

Die Erfinder haben nun völlig überraschend festgestellt, daß die mindestens zweifache kombinierte Expression von PSI, AFP, ChiS, ChiG oder GluG bezüglich der erworbenen Pilzresistenz bei transgenen Pflanzen zu synergistischen Effekten führt. Insbesondere werden die Wirkungen der Einzelsubstanzen in der Kombination deutlich übertroffen. Hierzu gehören die Resistenz gegen den Pilz Rhizoctonia solani, Sclerotinia-Befall, Botrytis-Befall usw.The inventors have now found, completely surprisingly, that the at least two-fold combined expression of PSI, AFP, ChiS, ChiG or GluG leads to synergistic effects in transgenic plants with regard to the acquired fungal resistance. In particular, the effects of the individual substances in the combination are clearly exceeded. These include resistance to the fungus Rhizoctonia solani, Sclerotinia infestation, Botrytis infestation, etc.

Erfindungsgemäße Kombinationen sind (DNA und/oder Polypeptide):Combinations according to the invention are (DNA and / or polypeptides): (Zweierkombinationen)(Combinations of two)

Figure imgb0001
Figure imgb0001

(Dreierkombinationen)(Combinations of three)

Figure imgb0002
Figure imgb0002

(Viererkombinationen)(Combinations of four)

ChiS, GluG, PSI, AFP; ChiS, GluG, PSI, ChiG;ChiS, GluG, PSI, AFP; ChiS, GluG, PSI, ChiG;

(Fünferkombination)(Combination of five)

Chis, GluG, PSI, AFP, ChiG
Chis, GluG, PSI, AFP, ChiG

Gegenstand der Erfindung ist weiterhin die kombinierte Verwendung der Proteine mit pathogen-inhibierender Wirkung, vorzugsweise ChiS, PSI, AFP, ChiG und GluG, gegen Pathogene. Kombinierte Verwendung bedeutet hier auch, daß mindestens eine erste pathogene inhibierende Substanz von dem Organismus exprimiert und mindestens eine zweite Substanz, die pathogen inhibierende Wirkung hat, von außen auf den Organismus aufgebracht wird.The invention further relates to the combined use of the proteins with pathogen-inhibiting activity, preferably ChiS, PSI, AFP, ChiG and GluG, against pathogens. Combined use here also means that at least one first pathogenic inhibiting substance is expressed by the organism and at least one second substance which has a pathogenic inhibiting effect is applied to the organism from the outside.

Zu den erfindungsgemäßen Mitteln zählen auch jene, die die oben genannten Proteine in mindestens zweifacher Kombination enthalten. Die erfindungsgemäßen Mittel können neben den Proteinen weitere Wirkstoffe enthalten. Diese weiteren Wirkstoffe können Pestizide, Düngemittel und/oder Wachstumsregulatoren sein, die erfindungsgemäßen Mittel können zudem in unterschiedlichen Formulierungen bereitgestellt werden, wie Konzentrate, Emulsionen, Pulver, Formulierungen auf Trägerstoffen, Mischungen mit anderen Wirkstoffen, etc. Besonders bevorzugt wird die Kombination ChiS/PSI und AFP/PSI. Diese Proteine können besonders wirksam zur Wachstumshemmung von Rhizoctonia solani, insbesondere bei Tabakpflanzenkulturen, eingesetzt werden.The agents according to the invention also include those which contain the abovementioned proteins in at least two combinations. In addition to the proteins, the agents according to the invention can contain further active ingredients. These further active ingredients can be pesticides, fertilizers and / or growth regulators, the agents according to the invention can also be provided in different formulations, such as concentrates, emulsions, powders, formulations on carriers, mixtures with other active ingredients, etc. The combination ChiS / PSI is particularly preferred and AFP / PSI. These proteins can be used particularly effectively to inhibit the growth of Rhizoctonia solani, in particular in tobacco plant crops.

Gegenstand der Erfindung ist auch die Verwendung einer DNA-Sequenz in einem erfindungsgemäßen Verfahren, die mindestens für ein Polypeptid der Sequenzen A bis E kodiert, bzw. ein pathogen-resistenter Organismus, wobei dessen Genom mindestens zwei unterschiedliche Gene unter der Kontrolle aktiver Promotoren mit pathogen-inhibierender Wirkung enthält, wobei die Gene jeweils aus der Gruppe der Sequenzen A bis E ausgewählt sind. Die Erfindung schließt weiterhin DNA-Sequenzen ein, die mit einer DNA-Sequenz hybridisiert, welche für Polypeptide der Aminosäuresequenzen A bis E kodiert, wobei diese DNA-Sequenzen natürlichen synthetischen oder halbsynthetischen Ursprungs sein kann und mit der zuvor genannten DNA-Sequenz durch Mutationen, Nukleotidsubstitutionen, Nukleotiddeletionen, Nukleotidinsertionen und Inversionen von Nukleotidfolgen verwandt sein kann und für ein Polypeptid mit pathogener Wirksamkeit kodiert. Gegenstand der Erfindung ist weiter noch ein rekombinantes DNA-Molekül, welches mindestens eine DNA-Sequenz nach den vorstehenden Ausführungen enthält, wobei dieses DNA-Molekül als Klonierungs- oder Expressionsvektor vorliegen kann.The invention also relates to the use of a DNA sequence in a method according to the invention which codes at least for a polypeptide of the sequences A to E, or a pathogen-resistant organism, the genome of which has at least two different genes under the control of active promoters with pathogen contains an inhibitory effect, the genes each being selected from the group of sequences A to E. The invention further includes DNA sequences which hybridize with a DNA sequence which codes for polypeptides of the amino acid sequences A to E, which DNA sequences can be of natural synthetic or semi-synthetic origin and with the aforementioned DNA sequence by mutations, Nucleotide substitutions, nucleotide deletions, Nucleotide insertions and inversions of nucleotide sequences can be related and encoded for a polypeptide with pathogenic activity. The invention furthermore relates to a recombinant DNA molecule which contains at least one DNA sequence as described above, it being possible for this DNA molecule to be present as a cloning or expression vector.

Gegenstand der Erfindung sind entsprechende Wirtsorganismen und Zwischenwirte, die mit einem rekombinanten DNA-Molekül nach den vorstehenden Ausführungen transformiert sind. Als Zwischenwirt bei der Erzeugung eines pathogen-resistenten transgenen Organismus werden Bakerienstämme bevorzugt, insbesondere sogenannte Agrobakerienstämme.The invention relates to corresponding host organisms and intermediate hosts which have been transformed with a recombinant DNA molecule as described above. Bakery strains are preferred as intermediate hosts in the production of a pathogen-resistant transgenic organism, in particular so-called agrobacterial strains.

Gegenstand der Erfindung sind weiterhin die nach dem erfindungsgemäßen Verfahren erhaltenen transgenen pathogen-resistenten Organismen, insbesondere Tabak-, Kartoffel-, Mais-, Erbsen-, Raps- und Tomatenpflanzen.The invention further relates to the transgenic pathogen-resistant organisms obtained by the process according to the invention, in particular tobacco, potato, corn, pea, rape and tomato plants.

Die erfindungsgemäßen DNA-Sequenzen werden in der Regel zusammen mit einem Promotor transferiert. Promotorsequenzen werden vom pflanzlichen Transskriptionsapparat erkannt und führen somit zu einer konstitutiven Expression des mit ihnen verbundenen Gens in Pflanzen. Der Promotor kann aber auch Pathogen-induzierbar und/oder verwundungs-induzierbar (WUN1) und/oder gewebespezifisch und/oder entwicklungsspezifisch sein.The DNA sequences according to the invention are generally transferred together with a promoter. Promoter sequences are recognized by the plant transcription apparatus and thus lead to a constitutive expression of the gene associated with them in plants. However, the promoter can also be pathogen-inducible and / or wound-inducible (WUN1) and / or tissue-specific and / or development-specific.

Die zur Durchführung der Erfindung erforderlichen gentechnologischen Arbeiten, insbesondere zur Expression des Gens in Pflanzen, sind allgemein bekannt. Beispielsweise aus der Veröffentlichung von Maniatis et al. in "Molecular cloning: A laboratory manual", Cold Spring Harbour (1982)
Die Erfindung wird in den folgenden Beispielen näher erläutert.The genetic engineering work required to carry out the invention, in particular for the expression of the gene in plants, is generally known. For example, from the publication by Maniatis et al. in "Molecular cloning: A laboratory manual", Cold Spring Harbor (1982)
The invention is explained in more detail in the following examples.

Alle mokekularbiologischen Standard-Methoden wurden, sofern nicht anders angegeben, wie bei Maniatis et al. "Molecular cloning: a laboratory manual", Cold Spring Harbour, (1982) beschrieben, durchgeführt.Unless otherwise stated, all standard biological biological methods were used as described in Maniatis et al. "Molecular cloning: a laboratory manual", Cold Spring Harbor, (1982).

Die für die Aminosäuresequenzen A bis E kodierende DNA wurde zunächst in an sich bekannter Weise kloniert und dann durch Konjugation nach A. Tumefaciens LBA 4404 (A. Hoekema et al., Nature 303, 179-180) transferiert. Dies geschah nach der von Van Haute et al. in EMBO J. 2, 411-418 (1983), beschriebenen Methode.The DNA coding for the amino acid sequences A to E was first cloned in a manner known per se and then transferred by conjugation to A. Tumefaciens LBA 4404 (A. Hoekema et al., Nature 303 , 179-180). This was done according to the Van Haute et al. method described in EMBO J. 2 , 411-418 (1983).

Die Überprüfung der DNA-Transfers in das Agrobakterium erfolgte durch die Isolierung von Agrobakterien-DNA nach der von Ebert et al. in Proc. Natl. Acad. Sci. USA 84 5745-5749 (1987), geschilderten Methode. Die Restriktionsspaltung der DNA, der Transfer auf Hybond-N-Membran (Amersham) und die Hybridisierung gegen eine radioaktiv markierte DNA-Sonde gaben Aufschluß über einen erfolgreichen DNA-Transfer in das Agrobakterium.The DNA transfers into the agrobacterium were checked by isolating agrobacterial DNA according to the method described by Ebert et al. in proc. Natl. Acad. Sci. USA 84 5745-5749 (1987), method described. The restriction cleavage of the DNA, the transfer to Hybond-N membrane (Amersham) and the hybridization against a radioactively labeled DNA probe provided information about a successful DNA transfer into the agrobacterium.

Mittels des transformierten Agrobakteriums wurden wiederum Tabak-, Raps-, Erdbeer-, Tomaten- und Kartoffelpflanzen transformiert.Tobacco, rapeseed, strawberry, tomato and potato plants were in turn transformed using the transformed agrobacterium.

Die zur Infektion benötigten Agrobakterien LBA 4404 wurden in selektivem Antibiotika-Medium angezogen (P. Zambrisky et al. in EMBO J., 1, 147-152 (1983)), durch Zentrifugation sedimentiert und in YEB-Medium ohne Antibiotika gewaschen (YEB = 0,5% Fleisch-Extrakt; 0,2% Hefeextrakt; 0,5% Pepton; 0,5% Saccharose; 2mM MgSO₄). Nach erneuter Sedimentation und Aufnahme in MgSO₄ konnten die Bakterien zur Infektion verwendet werden.The LBA 4404 agrobacteria required for infection were grown in selective antibiotic medium (P. Zambrisky et al. In EMBO J., 1 , 147-152 (1983)), sedimented by centrifugation and washed in YEB medium without antibiotics (YEB = 0.5% meat extract; 0.2% yeast extract; 0.5% peptone; 0.5% sucrose; 2mM MgSO₄). After renewed sedimentation and absorption in MgSO₄, the bacteria could be used for infection.

Zur Infektion wurde die sogenannte Blattscheibenmethode eingesetzt.The so-called leaf disc method was used for infection.

Für die Blattscheiben-Infektion wurden sterile Blätter verwendet. Etwa 1 cm große Blattstücke werden in die zuvor beschriebene Agrobakteriensuspension eingetaucht und anschließend auf 3MS-Medium überführt (Medium nach T. Murashige und F. Skoog in Physiol. Plant., 15, 473-497 (1962); 3MS = MS + 3% Saccharose). Nach zweitägiger Inkubation bei 16 Stunden Licht und 25°C bis 27°C wurden die Blattstücke auf MSC16-Medium (nach T. Murashige (siehe oben); MSC16 = MS + 0,5 µg/ml BAP + 0,1 µg/ml NAA + 100 µg/ml Kanamycinsulfat + 500 µg/ml Claforan) überführt. Nach 4-6 Wochen erscheinende Sprosse wurden abgeschnitten und auf MSC15-Medium (nach Murashige (siehe oben); MSC15 = MS + 2% Saccharose, 500 µg/ml Claforan + 100 µg/ml Kanamycinsulfat) umgesetzt. Sprosse mit Wurzelbildung wurden weiter analysiert.Sterile leaves were used for leaf disc infection. Leaf pieces of about 1 cm in size are immersed in the agrobacterial suspension described above and then transferred to 3MS medium (medium according to T. Murashige and F. Skoog in Physiol. Plant., 15 , 473-497 (1962); 3MS = MS + 3% Sucrose). After two days of incubation in 16 hours of light and 25 ° C to 27 ° C, the leaf pieces were placed on MSC16 medium (according to T. Murashige (see above); MSC16 = MS + 0.5 µg / ml BAP + 0.1 µg / ml NAA + 100 µg / ml kanamycin sulfate + 500 µg / ml Claforan) transferred. Shoots appearing after 4-6 weeks were cut off and reacted on MSC15 medium (according to Murashige (see above); MSC15 = MS + 2% sucrose, 500 µg / ml claforan + 100 µg / ml kanamycin sulfate). Root-shaped shoots were further analyzed.

Monokotyledone Pflanzen (u. a. Mais), zum Teil aber auch dikolyte Pflanzen wurden mittels direktem Gentransfer in Protoplasten transformiert. Diese Protoplasten wurden anschließend zu intakten Pflanzen regeneriert (Beispiel: J. Potrykus in Biotechnology 8 (1990) 535).Monocotyledonous plants (including maize), but also some diclyte plants, were transformed into protoplasts using direct gene transfer. These protoplasts were then regenerated to intact plants (example: J. Potrykus in Biotechnology 8 (1990) 535).

Die erhaltenen transgenen Pflanzen wurden zu Testzwecken mit dem Pilz Rhizoctonia solani infiziert. Hierzu wurden Pilzkulturen gezüchtet und in Einheitserde gründlich vermischt. Diese Erde wurde dann in einer Schale verteilt und mit den zu testenden Pflanzen bepflanzt.The transgenic plants obtained were infected with the fungus Rhizoctonia solani for test purposes. For this purpose, mushroom cultures were grown and mixed thoroughly in common soil. This soil was then distributed in a bowl and planted with the plants to be tested.

Zur Auswertung wurde jeder Pflanze einer Schale ein Wert von 0 bis 3 zugeordnet. Daraus konnte für jede Pflanzenlinie ein Index berechnet werden, der sich aus der Summe der Werte ergab. Die Einteilung ist wie folgt:

0 =
Ohne Symptome (gesund)
1 =
leicht reduzierte Größe (gegenüber einer nicht-infizierten Kontrolle); kein bis sehr geringer sichtbarer Befall
2 =
starke Wachstumsreduktion; schwere Befallssymptome
3 =
tot
Die Bewertung erfolgt jeweils 14 Tage nach Start der Versuchsreihe.Each plant was assigned a value from 0 to 3 for evaluation. From this, an index could be calculated for each plant line, which resulted from the sum of the values. The division is as follows:
0 =
Without symptoms (healthy)
1 =
slightly reduced size (compared to a non-infected control); no or very little visible infestation
2 =
strong growth reduction; severe symptoms of infestation
3 =
dead
The evaluation takes place 14 days after the start of the test series.

Beispiel 1: Example 1 : Pilzinhibitionstest mit kombinierten ProteinenMushroom inhibition test with combined proteins

Es sollte zunächst einmal gezeigt werden, daß die hier verwendeten Proteine in ihrer Kombination synergistische Wirkungen haben. Hierzu wurden in vitro Pilzwachstumstests durchgeführt.First of all it should be shown that the proteins used here have synergistic effects in their combination. For this purpose, fungal growth tests were carried out in vitro.

Hierbei wurde eine definierte Menge an Rhizoctonia solani Pilzmycel mit 100 µl-Kartoffel-Dextroselösung versetzt und in Mikrotiterplatten bei 25°C inkubiert. Dabei korreliert das Wachstum des Pilzes mit der Zunahme der optischen Dichte bei 405 Nanometer linear. Die inhibierende Wirkung von Proteinen kann anhand eines geringeren Anstiegs der optischen Dichte nachgewiesen werden.A defined amount of Rhizoctonia solani mushroom mycelium was mixed with 100 µl potato dextrose solution and incubated in microtiter plates at 25 ° C. The growth of the fungus correlates linearly with the increase in optical density at 405 nanometers. The inhibitory effect of proteins can be demonstrated by a smaller increase in optical density.

Aus einer Flüssigkultur von R. Solani wurden 2-3 Mycelbällchen entnommen, in einem Eppendorfgefäß mit 100 µl KGB-Medium versetzt und mit einem Glasmörser vorsichtig homogenisiert. Diese Suspension wurde dann mit 10 ml KGB-Medium gemischt und durch ein steriles 100 µm Sieb gegeben. Die optische Dichte dieser Mycelfragment-Suspension (100 µl-Aliquot) wurde durch Zugabe von Medium auf einen Wert von 0,06-0,07 bei 405 Nanometer eingestellt. Je 100 µl wurden auf eine Mikrotiterplatte gegeben und mit den zu testenden Proteinen versetzt. Pro Ansatz wurden 7 Parallelen gemessen. Als Kontrolle dienen Ansätze, die mit den entsprechenden Mengen an Puffer versetzt wurden. Die Platten wurden über 48 Stunden bei 25°C im Dunkeln inkubiert und die optische Dichte der Kulturen in regelmäßigen Abständen gemessen.2-3 mycelium balls were removed from a liquid culture from R. Solani, mixed with 100 µl KGB medium in an Eppendorf tube and carefully homogenized with a glass mortar. This suspension was then mixed with 10 ml KGB medium and passed through a sterile 100 µm sieve. The optical density of this mycelium fragment suspension (100 μl aliquot) was adjusted to a value of 0.06-0.07 at 405 nanometers by adding medium. 100 µl each were placed on a microtiter plate and mixed with the proteins to be tested. 7 parallels were measured per approach. Approaches which have been mixed with the appropriate amounts of buffer serve as a control. The plates were incubated for 48 hours at 25 ° C in the dark and the optical density of the cultures was measured at regular intervals.

Ob zwei Proteine bei der Wachstumshemmung des Pilzes in additiver synergistischer oder antagonistischer Weise zusammenwirken, läßt sich aus den gemessenen Daten mit Hilfe der im folgenden beschriebenen und allgemeinen angewandten Colby-Formel errechnen (S. R. Colby in Wheeds, 15 (1967), 20-22).Whether two proteins interact in an additive synergistic or antagonistic manner in the growth inhibition of the fungus can be calculated from the measured data with the aid of the Colby formula described and generally used below (SR Colby in Wheeds, 15 (1967), 20-22) .

Hierzu war es zunächst notwendig, die bei einem additiven Verhalten theoretisch zu erwartende Wachstumshemmung E (der erwartete Wirkungsgrad) zu berechnen. Dieser ist gegeben durch:

E = W1 + W2 - ((W1 X W2)/100)

Figure imgb0003


Dabei geben W1 und W2 die Wirkungsgrade der einzelnen Proteine an, worunter man die prozentuale Abweichung der Wachstumskurve (in Anwesenheit des Proteins) von der unbehandelten Kontrolle versteht. Der Wirkungsgrad für ein Protein ist (zu einem bestimmten Zeitpunkt der Wachstumskurve) gegeben durch:

W1 = (OD(K) - OD(P))/OD(K) X 100   (Prozent)
Figure imgb0004

Hierbei ist OD(K) die optische Dichte der unbehandelten Kontrolle und OD(P) die optische Dichte der mit dem Protein behandelten Kultur.To do this, it was first necessary to calculate the growth inhibition E (the expected efficiency) that could theoretically be expected for an additive behavior. This is given by:

E = W1 + W2 - ((W1 X W2) / 100)
Figure imgb0003

W1 and W2 indicate the efficiencies of the individual proteins, which means the percentage deviation of the growth curve (in the presence of the protein) from the untreated control. The efficiency for a protein is given (at a certain point in the growth curve) by:

W1 = (OD (K) - OD (P)) / OD (K) X 100 (percent)
Figure imgb0004

Here, OD (K) is the optical density of the untreated control and OD (P) is the optical density of the culture treated with the protein.

Bei der kombinierten Anwendung von zwei Proteinen waren somit folgende Aussagen möglich: Ist der im Experiment gemessene Wirkungsgrad G gleich dem Erwartungswert E, so handelt es sich um ein additives Verhalten. Ist G hingegen größer als E, so liegt synergistisches Verhalten vor.When using two proteins in combination, the following statements were possible: Is the one measured in the experiment Efficiency G equal to the expected value E, it is an additive behavior. If, on the other hand, G is greater than E, there is synergistic behavior.

Unter Verwendung dieses Prüfmodells ergaben sich für die im Beispiel verwendeten Proteine ChiS, PSI, AFP, ChiG und GluG überraschenderweise synergistische Hemmeffekte gegen diverse Pilze, wobei diese Effekte sowohl durch die Kombination zweier Proteinarten, als auch durch die Mehrfachkombination der obengenannten Proteine erreicht wurde.Using this test model, the proteins ChiS, PSI, AFP, ChiG and GluG used in the example surprisingly showed synergistic inhibitory effects against various fungi, these effects being achieved both by the combination of two types of protein and by the multiple combination of the above-mentioned proteins.

Beispielsweise wurde aus der Kombination von ChiS und PSI-Protein, bzw. aus der Kombination von AFP- und PSI-Protein gegen den Pilz Rhizoctonia solani folgende Werte ermittelt (je zwei verschiedene ChiS und AFP-Konzentrationen bei konstanter RIP-Konzentration):For example, the following values were determined from the combination of ChiS and PSI protein or from the combination of AFP and PSI protein against the fungus Rhizoctonia solani (two different ChiS and AFP concentrations at a constant RIP concentration):

ChiS + PSI:ChiS + PSI:

Die Erwartungswerte waren:
E1 = 29,9% und E2 = 44,5%
Die gemessenen Werte waren:
G1 = 60,4% und G2 = 64,1%

Die Proteine ChiS und PSI wirken also bei der Wachstumshemmung von R. Solani in synergistischer Weise zusammen.
The expected values were:
E1 = 29.9% and E2 = 44.5%
The measured values were:
G1 = 60.4% and G2 = 64.1%

The proteins ChiS and PSI therefore work together in a synergistic manner in the inhibition of growth by R. Solani.

Die Fig. 1 zeigt die Ergebnisse, die mit der Kombination der Proteine, als auch mit den Einzelsubstanzen erhalten wurden. Nach der Figur werden verschiedene ChiS-Konzentrationen (0,5 µg/ml bzw. 0,05 µg/ml) mit PSi-Protein (1,0 µg/ml) kombiniert.Fig. 1 shows the results obtained with the combination of the proteins as well as with the individual substances. According to the figure, different ChiS concentrations (0.5 µg / ml or 0.05 µg / ml) are combined with PSi protein (1.0 µg / ml).

AFP + PSI:AFP + PSI:

Die Erwartungswerte waren:
E1 = 39,9% und E2 = 41,9%
Die gemessenen Werte waren:
G1 = 57,7% und G2 = 65,4%

Auch die Kombination AFP und PSI zeigt demnach eine synergistische Wachstumshemmung des Pilzes R. Solani an. In der Fig. 2 werden die Testergebnisse bei verschiedenen AFP-Konzentrationen (0,4 µg/ml bzw. 0,04 µg/ml) mit PSI-Protein (1,0 µg/ml) kombiniert angegeben.
The expected values were:
E1 = 39.9% and E2 = 41.9%
The measured values were:
G1 = 57.7% and G2 = 65.4%

The combination of AFP and PSI also indicates a synergistic growth inhibition of the R. Solani mushroom. 2 shows the test results at various AFP concentrations (0.4 µg / ml or 0.04 µg / ml) combined with PSI protein (1.0 µg / ml).

Beispiel 2:Example 2: Transgene PflanzenTransgenic plants

Um die erfindungsgemäßen Organismen mit synergistisch zusammenwirkenden DNA-Sequenzen zu erhalten, wurden zunächst transgene Pflanzen erzeugt, die mindestens eines der synergistisch zusammenwirkenden Gene enthielten.In order to obtain the organisms according to the invention with DNA sequences interacting synergistically, transgenic plants were first generated which contained at least one of the genes interacting synergistically.

ChiS in transgenen PflanzenChiS in transgenic plants

Es wurde zunächst ein ChiS-Gen mit pflanzlichen Regulations-sequenzen fusioniert.A ChiS gene was first fused with plant regulatory sequences.

Ein 1,8 Kb großes ChiS-Gen wurde durch die Verwendung von synthetischen Oligonukleotiden nach der Dideoxy-sequenzierungsmethode von Sanger et al. in Proc. Natl. Acad. Sci. USA, 74, (1977), 5463-5467, sequenziert.A 1.8 Kb ChiS gene was generated using synthetic oligonucleotides according to the dideoxy sequencing method of Sanger et al. in proc. Natl. Acad. Sci. USA, 74 , (1977), 5463-5467.

Der aus dem Blumenkohlmosaikvirus (CamV) stammende 35S-Promotor (400 bp (nach Töpfer et al. in Nucl. Acid. Res., 15 (1987) 5890)) wurde transskriptionell mit dem ChiS-Gen fusioniert. 3' vom ChiS-Gen wurde das 0,2 Kb große Terminationssignal des 35S-Gens des CamV verwendet, dessen Funktionalität in dikotylen Pflanzen bekannt ist. Das chimäre Gen 35S-ChiS wurde in den Vektor pLS034 kloniert, mittels des Agrobakteriums tumefaciens-Transformationssystems in Tabak- und Kartoffelpflanzen transformiert und Kanamycin-resistente Pflanzen regeneriert.The 35S promoter (400 bp (according to Töpfer et al. In Nucl. Acid. Res., 15 (1987) 5890)) derived from the cauliflower mosaic virus (CamV) was transcriptionally fused with the ChiS gene. 3 'from the ChiS gene the 0.2 Kb termination signal of the 35S gene of the CamV was used, the functionality of which is known in dicotyledonous plants. The chimeric gene 35S-ChiS was cloned into the vector pLS034, transformed into tobacco and potato plants by means of the Agrobacterium tumefaciens transformation system, and kanamycin-resistant plants were regenerated.

In den erhaltenen Pflanzen konnte sowohl das ChiS-Gen als auch die entsprechende mRNA sowie das Genproduktprotein nachgewiesen werden.Both the ChiS gene and the corresponding mRNA and the gene product protein could be detected in the plants obtained.

PSI in transgenen PflanzenPSI in transgenic plants

Aus reifen Gerstesamen (Hordeum vulgare L. cv. Piggy) wurde zunächst PolyA⁺⁻ RNA isoliert und in einer cDNA-Genbank in λ-gt-11-Phagen abgelegt. Die Einzelheiten des Verfahrens sind R. Lea in Plant. Biol., 12 (1989), 673-682, zu entnehmen. Mit Hilfe monospezifischer PSI-Antikörper wurden dann cDNA-Klone identifiziert.PolyA⁺⁻ RNA was first isolated from ripe barley seeds (Hordeum vulgare L. cv. Piggy) and stored in a cDNA library in λ-gt-11 phages. The details of the procedure are R. Lea in Plant. Biol., 12 (1989), 673-682. Monospecific PSI antibodies were then used to identify cDNA clones.

Im Anschluß daran wurden die PSI-positiven λ-gt-11-Phagen isoliert, weiter kloniert und nach der oben angegebenen Dideoxy-sequenzierungsmethode von Sanger et al. sequenziert. Die in E. Coli geklonte DNA wurde dann in der oben beschriebenen Weise durch Konjugation auf das Agrobakterium tumefaciens LBA4404 übertragen.Subsequently, the PSI-positive λ-gt-11 phages were isolated, further cloned and according to the dideoxy sequencing method of Sanger et al. sequenced. The DNA cloned in E. Coli was then conjugated to Agrobacterium tumefaciens LBA4404 in the manner described above.

Sowohl das transferierte Gen als auch mRNA und Genprodukt konnten in entsprechenden transgenen Tabak-, Kartoffel-, Raps-, Erdbeer- und Tomatenpflanzen nachgewiesen werden.The transferred gene as well as mRNA and gene product could be detected in corresponding transgenic tobacco, potato, rapeseed, strawberry and tomato plants.

AFP in transgenen PflanzenAFP in transgenic plants

Die cDNA-Sequenz des antifungischen Peptids wird für die Klonierung im Vektor mit Enden versehen, die in BamH1- und Sal1-Restriktionsschnittstellen ligiert werden können. Als Klonierungsvektor wurde pDH51 (Pietrzak et al. in Nucl. Acids Res. 14 (1986), 5857) verwendet. Der Vektor pDH51 wurde mit den Restriktionsenzymen BamH1 und Sal1 zwischen Promotor und Terminator geöffnet. Der Vektor pDH51 ist ein pUC18-Derivat, das Promotor- und Terminatorsequenzen des 35S-Transkripts aus Blumenkohlmosaikvirus enthält. Diese Sequenzen werden vom pflanzlichen Transskriptionsapparat erkannt und führen zu einer starken konstitutiven Expression des mit ihnen verbundenen Gens in Plfanzen. Die DNA des antifungischen Peptids wird dann über die BamH1 und Sal1-Schnittstelle in den Vektor kloniert. Schließlich wird die Transskriptionseinheit - Promotor, Gen und Terminator - mit dem Restriktionsenzym EcoRI aus dem Vektor herausgeschnitten und in einen Pflanzentransformationsvektor kloniert. Als Pflanzentransformationsvektor können zum Beispiel folgende Vektoren bzw. ihre Derivate verwendet werden:
   pOCA18 (Olszewski et al. in Nucl. Acids Res., 16 (1988), 10765) pPCV310 (Koncz und Shell in MGG 204 (1986), 383) und pBin19 (Bevan et al. Nucl. Acids. Res. 12 (1984) 8711)
Nachdem die Transskriptionseinheit und der Vektor über die EcoRI-Schnittstelle ligiert wurde, wurde das Konstrukt in den Agrobakterienstamm MP90RK (Koncz und Shell (siehe oben)) oder IHA101 (Hood et al. in J. Bacteriol. 168 (1986), 1291) konjugiert.The cDNA sequence of the antifungal peptide is terminated for cloning in the vector which can be ligated into BamH1 and Sal1 restriction sites. PDH51 (Pietrzak et al. In Nucl. Acids Res. 14 (1986), 5857) was used as the cloning vector. The vector pDH51 was opened with the restriction enzymes BamH1 and Sal1 between the promoter and terminator. The vector pDH51 is a pUC18 derivative, the promoter and terminator sequences of the 35S transcript Contains cauliflower mosaic virus. These sequences are recognized by the plant transcription apparatus and lead to a strong constitutive expression of the gene associated with them in plants. The DNA of the antifungal peptide is then cloned into the vector via the BamH1 and Sal1 cleavage site. Finally, the transcription unit - promoter, gene and terminator - is cut out of the vector with the restriction enzyme EcoRI and cloned into a plant transformation vector. The following vectors or their derivatives, for example, can be used as the plant transformation vector:
pOCA18 (Olszewski et al. in Nucl. Acids Res., 16 (1988), 10765) pPCV310 (Koncz and Shell in MGG 204 (1986), 383) and pBin19 (Bevan et al. Nucl. Acids. Res. 12 (1984 ) 8711)
After the transcription unit and the vector were ligated via the EcoRI site, the construct was conjugated into the agrobacterial strain MP90RK (Koncz and Shell (see above)) or IHA101 (Hood et al. In J. Bacteriol. 168 (1986), 1291) .

Transgene Tabak-, Kartoffel-, Erdbeer-, Raps- und Tomatenpflanzen wurden dann nach der oben beschriebenen Methode transformiert. Transformierte Sprosse werden aufgrund der mitübertragenen Resistenz gegen das Antibiotikum Kanamycin selektioniert. Durch DNA-Analyse (Southern Blotting), RNA-Analyse (Northern Blotting) und Proteinanalyse mit spezifischen Antikörpern (Western Blotting) wurde die Expression des antifungischen Proteins in den transformierten Nutzpflanzen überprüft und bestätigt.Transgenic tobacco, potato, strawberry, rapeseed and tomato plants were then transformed using the method described above. Transformed shoots are selected for their resistance to the antibiotic kanamycin. The expression of the antifungal protein in the transformed useful plants was checked and confirmed by DNA analysis (Southern blotting), RNA analysis (Northern blotting) and protein analysis with specific antibodies (Western blotting).

ChiG und GluG in transgenen PflanzenChiG and GluG in transgenic plants

Analog zu den zuvor beschriebenen Pflanzen konnten ChiG- bzw. GluG-transgene Pflanzen erhalten werden, die sowohl Southern-, Northern- als auch Western-positiv waren.Analogous to the plants described above, ChiG or GluG transgenic plants were obtained which were both Southern, Northern and Western positive.

ChiS, PSI, AFP, ChiG, GluG in transgenen monokotylen PflanzenChiS, PSI, AFP, ChiG, GluG in transgenic monocotyledonous plants

Die zuvor genannten Gene konnten mittels direktem Gentransfer in das Genom monokotyler Pflanzen wie beispielsweise Mais integriert werden. Hierbei wurden transgene Pflanzen erhalten, die sowohl Southern- als auch Northern- und Western-positiv waren.The aforementioned genes could be integrated into the genome of monocotyledonous plants such as maize by means of direct gene transfer. In this way, transgenic plants were obtained which were both Southern, Northern and Western positive.

Kombination verschiedener Pilzresistenzgene in transgenen PflanzenCombination of different fungal resistance genes in transgenic plants

Die zuvor erhaltenen Tabak-, Mais-, Raps-, Erdbeer-, Kartoffel- und Tomatenpflanzen wurden miteinander gekreuzt und auf Pflanzen selektioniert, die jeweils die Pilzresistenzgene beider Eltern beeinhalteten. Darüberhinaus wurden transgene Pflanzen dadurch erhalten, daß sie zunächst mit einem und dann mit einem oder mehreren weiteren Gen transformiert wurden. Schließlich wurden auch noch Pflanzen mit Vektoren transformiert, die verschiedene Resistenzgene beeinhalteten. Mit diesem Pflanzengut wurden Pilzresistenztests gemacht. Überraschenderweise sind in allen Fällen nicht nur additive Effekte bezüglich der Pilzresistenz zu beobachten, sondern synergistische Effekte.The tobacco, maize, rapeseed, strawberry, potato and tomato plants obtained previously were crossed with one another and selected on plants which in each case contained the fungal resistance genes of both parents. In addition, transgenic plants were obtained by first transforming them with one and then with one or more other genes. Finally, plants were transformed with vectors that contained various resistance genes. Fungus resistance tests were carried out with this plant material. Surprisingly, not only additive effects regarding fungus resistance can be observed in all cases, but synergistic effects.

Beispielsweise zeigt eine Tabakpflanze, die ChiS und PSI exprimiert, eine wesentlich stärkere Widerstandsfähigkeit gegen Rhizoctonia-Befall als die Pflanzen, die entweder nur ChiS oder PSI exprimierten, oder die sich aus der additiven Widerstandsfähigkeit ergeben würde.For example, a tobacco plant that expresses ChiS and PSI shows a much greater resistance to Rhizoctonia attack than the plants that either only expressed ChiS or PSI or that would result from the additive resistance.

Ein synergistischer Hemmeffekt ergibt sich auch aus der kombinierten Expression von PSI- und AFP-transgenen Tabak gegen Rhizoctonia solani Befall. Auch die zwei- oder mehrfache Kombination verschiedener Gene (ChiS, RIP, AFP, ChiG und GluG) in den unterschiedlichsten transgenen Pflanzen führte zu synergistischen Hemmeffekten gegen diverse Pilze.A synergistic inhibitory effect also results from the combined expression of PSI and AFP transgenic tobacco against Rhizoctonia solani infestation. The two or more combinations of different genes (ChiS, RIP, AFP, ChiG and GluG) in a wide variety of transgenic plants also led to synergistic inhibitory effects against various fungi.

Während Wildtyppflanzen bei Tests mit 20 Sämlingen Indexwerte von 38 bis 46 aufweisen, erweist sich bei erfindungsgemäßen transgenem Tabak, daß dieser in Anwesenheit des Pilzes Rhizoctonia solani so gut wächst wie Kontrollpflanzen (Indexwert 10-12), die auf Rhizoctonia-freiem Boden kultiviert wurden.

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While wild type plants have index values of 38 to 46 in tests with 20 seedlings, it has been found in transgenic tobacco according to the invention that in the presence of the fungus Rhizoctonia solani it grows as well as control plants (index value 10-12) which were cultivated on Rhizoctonia-free soil.
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Claims (12)

Transgener pathogen-resistenter Organismus
dadurch gekennzeichnet, daß sein Genom mindestens zwei unterschiedliche Gene unter Kontrolle aktiver Promotoren mit pathogen-inhibierender Wirkung enthält.Transgenic pathogen-resistant organism
characterized in that its genome contains at least two different genes under the control of active promoters with pathogen-inhibiting activity. Transgener pathogen-resistenter Organismus nach Anspruch 1,
dadurch gekennzeichnet, daß die Gene für Genprodukte codieren, die die Vitalität von Pilzen herabsetzen.Transgenic pathogen-resistant organism according to claim 1,
characterized in that the genes code for gene products which reduce the vitality of fungi. Transgener pathogen-resistenter Organismus nach Anspruch 1 oder 2,
dadurch gekennzeichnet, daß die Gene pilzlichen, bakteriellen, pflanzlichen, tierischen oder viralen Ursprungs sind.Transgenic pathogen-resistant organism according to claim 1 or 2,
characterized in that the genes are of fungal, bacterial, plant, animal or viral origin. Transgener pathogen-resistenter Organismus nach Anspruch 2 oder 3,
dadurch gekennzeichnet, daß die Genprodukte die Pilzresistenz fördernde Eigenschaften haben.Transgenic pathogen-resistant organism according to claim 2 or 3,
characterized in that the gene products have anti-fungal properties. Transgener pathogen-resistenter Organismus nach Anspruch 4, dadurch gekennzeichnet, daß die Genprodukte
   Chitinase (ChiS, ChiG), Glukanase (GluG), Proteinsynthese-Inhibitor (PSI) und antifungales Protein (AFP)
sind.Transgenic pathogen-resistant organism according to claim 4, characterized in that the gene products
Chitinase (ChiS, ChiG), Glucanase (GluG), Protein Synthesis Inhibitor (PSI) and Antifungal Protein (AFP)
are. Transgener pathogen-resistenter Organismus nach einem der Ansprüche 1 bis 5,
dadurch gekennzeichnet, daß dieser eine Pflanze ist.Transgenic pathogen-resistant organism according to one of claims 1 to 5,
characterized in that it is a plant. Transgener pathogen-resistenter Organismus nach Anspruch 6, dadurch gekennzeichnet, daß es sich um eine Tabak-, Kartoffel-, Erdbeer-, Mais-, Raps- oder Tomatenpflanze handelt.Transgenic pathogen-resistant organism according to claim 6, characterized in that it is a tobacco, potato, strawberry, corn, rape or tomato plant. DNA-Übertragungsvektoren mit inserierten DNA-Sequenzen nach einem oder mehreren der vorstehenden Ansprüche.DNA transmission vectors with inserted DNA sequences according to one or more of the preceding claims. Verfahren zur Erzeugung pathogen-resistenter Organismen nach einem der Ansprüche 1-7,
dadurch gekennzeichnet, daß in das Genom eines Organismus mindestens ein Gen mit pathogen-inhibierender Wirkung transferiert und der pathogen-resistente Organismus a) durch Kreuzung des Organismus mit einem gegebenenfalls transgenen weiteren Organismus, der mindestens ein anderes Gen mit pathogen-inhibierender Wirkung enthält, und anschließende Selektion und/oder b) durch Transformation mindestens eines anderen Gens mit pathogen-inhibierender Wirkung in den Organismus erhalten wird.Method for producing pathogen-resistant organisms according to one of claims 1-7,
characterized in that at least one gene with a pathogen-inhibiting effect is transferred into the genome of an organism and the pathogen-resistant organism a) by crossing the organism with an optionally transgenic further organism which contains at least one other gene with a pathogen-inhibiting effect, and subsequent selection and / or b) by transforming at least one other gene with a pathogen-inhibiting effect into the organism is obtained. Verfahren nach Anspruch 9,
dadurch gekennzeichnet, daß DNA-Übertragungsvektoren mit inserierten DNA-Sequenzen entsprechend einem Gen mit pathogen-inhibierender Wirkung, wie in einem der Ansprüche 1 bis 5 beschrieben, verwendet werden.Method according to claim 9,
characterized in that DNA transmission vectors with inserted DNA sequences corresponding to a gene with a pathogen-inhibiting effect, as described in one of claims 1 to 5, are used. Verfahren zur Erzeugung pathogen resistenter Organismen nach einem der Ansprüche 1-7 ,
dadurch gekennzeichnet, daß zur Transformation in das Genom eines Organismus Vektoren verwendet werden, die mehr als ein Gen mit pathogen-inhibierender Wirkung umfassen.Method for producing pathogen-resistant organisms according to one of claims 1-7,
characterized in that vectors are used for the transformation into the genome of an organism which comprise more than one gene with pathogen-inhibiting action. Verfahren zur Gewährleistung der Resistenz von Organismen gegen Pathogene,
dadurch gekennzeichnet, daß als Organismus ein transgener pathogen-resistenter Organismus nach einem der Ansprüche 1 bis 7 oder ein Organismus, dessen Genom mindestens ein Gen gemäß der Definitionen der Ansprüche 1 bis 7 enthält, verwendet und auf den Organismus mindestens eine Substanz aufgebracht wird, die nicht durch den Organismus exprimiert wird, aber irgendeinem anderen der Genprodukte gemäß den Ansprüchen 1 bis 7 entspricht.Procedures for ensuring the resistance of organisms to pathogens,
characterized in that a transgenic pathogen-resistant organism according to one of claims 1 to 7 or an organism whose genome contains at least one gene as defined in claims 1 to 7 is used as the organism and at least one substance is applied to the organism which is not expressed by the organism, but corresponds to any other of the gene products according to claims 1 to 7.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018859A1 (en) * 1994-01-10 1995-07-13 Ciba-Geigy Ag Synergistic antifungal protein and compositions containing same
WO2000001846A2 (en) * 1998-07-03 2000-01-13 Devgen N.V. Characterisation of gene function using double stranded rna inhibition
US7087420B1 (en) 1997-07-17 2006-08-08 Cambia Microbial β-glucuronidase genes, gene products and uses thereof

Families Citing this family (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6414222B1 (en) * 1993-02-05 2002-07-02 Regents Of The University Of Minnesota Gene combinations for herbicide tolerance in corn
US6069298A (en) * 1993-02-05 2000-05-30 Regents Of The University Of Minnesota Methods and an acetyl CoA carboxylase gene for conferring herbicide tolerance and an alteration in oil content of plants
US6222099B1 (en) 1993-02-05 2001-04-24 Regents Of The University Of Minnesota Transgenic plants expressing maize acetyl COA carboxylase gene and method of altering oil content
US5530187A (en) * 1993-07-16 1996-06-25 The Salk Institute For Biological Studies Transgenic plants containing multiple disease resistance genes
DE4423022C1 (en) * 1994-06-30 1995-05-24 Lutz Prof Dr Heide Transgenic plants with increased content of resistance factor
US6740749B1 (en) * 1996-06-25 2004-05-25 Gsf-Forschungszentrum Fur Umwelt Ozone-induced gene expression in plants
JP4324656B2 (en) 1996-07-29 2009-09-02 ケイヘン ナームロゼ フェンノートシャップ Polynucleotides that modulate plant defense responses and uses thereof
US6121436A (en) 1996-12-13 2000-09-19 Monsanto Company Antifungal polypeptide and methods for controlling plant pathogenic fungi
WO2001027150A2 (en) 1999-10-14 2001-04-19 Clontech Laboratories Inc. Anthozoa derived chromo/fluoroproteins and methods for using the same
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US7417131B2 (en) * 2005-11-04 2008-08-26 Evrogen Joint Stock Company Modified green fluorescent proteins and methods for using same
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US8563703B2 (en) 2006-01-25 2013-10-22 Evrogen IP Joint Stock Company Fluorescent proteins and methods for using same
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US20100257634A1 (en) * 2009-04-03 2010-10-07 Venganza Inc. Bioassay for gene silencing constructs
CN101613685B (en) * 2009-05-20 2011-04-13 广东省昆虫研究所 Combined chitinase for deactivating varroa destructor and application thereof
RU2730038C2 (en) 2018-06-28 2020-08-14 Общество с ограниченной ответственностью "ПЛАНТА" Luciferin biosynthesis enzymes and use thereof

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989004371A1 (en) * 1987-11-02 1989-05-18 Louisiana State University Agricultural And Mechan Plants genetically enhanced for disease resistance
US4940840A (en) * 1984-03-26 1990-07-10 Dna Plant Technology Corporation Novel chitinase-producing bacteria and plants
US4970168A (en) * 1989-01-27 1990-11-13 Monsanto Company Virus-resistant plants
EP0440304A1 (en) * 1990-01-30 1991-08-07 Mogen International N.V. Fungal resistant plants, process for obtaining fungal resistant plants and recombinant polynucleotides for use therein
WO1991019738A1 (en) * 1990-06-15 1991-12-26 Hoechst Aktiengesellschaft Antifungal polypeptide, process for producing it
WO1992016632A1 (en) * 1991-03-25 1992-10-01 Elf Sanofi Recombinant dna coding for a novel protein having beta-1,3-glucanase activity of soya
WO1992017591A1 (en) * 1991-04-08 1992-10-15 Sandoz Ltd. A plant chitinase gene and use thereof
WO1994008009A1 (en) * 1992-10-05 1994-04-14 Mogen International N.V. Antifungal chitin binding proteins and dna coding therefor

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3810286A1 (en) * 1988-03-25 1989-10-12 Max Planck Gesellschaft TRANSGENIC PLANT WITH MODIFIED PHYSIOLOGY, MORPHOLOGY AND MODIFIED HORMONE METABOLISM, TISSUE CULTURES OF THIS PLANT AND METHOD FOR THE PRODUCTION THEREOF
DE4040954C2 (en) * 1990-12-20 2001-05-17 Max Planck Gesellschaft Process for the production of pathogen-resistant plants

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4940840A (en) * 1984-03-26 1990-07-10 Dna Plant Technology Corporation Novel chitinase-producing bacteria and plants
WO1989004371A1 (en) * 1987-11-02 1989-05-18 Louisiana State University Agricultural And Mechan Plants genetically enhanced for disease resistance
US4970168A (en) * 1989-01-27 1990-11-13 Monsanto Company Virus-resistant plants
EP0440304A1 (en) * 1990-01-30 1991-08-07 Mogen International N.V. Fungal resistant plants, process for obtaining fungal resistant plants and recombinant polynucleotides for use therein
WO1991019738A1 (en) * 1990-06-15 1991-12-26 Hoechst Aktiengesellschaft Antifungal polypeptide, process for producing it
WO1992016632A1 (en) * 1991-03-25 1992-10-01 Elf Sanofi Recombinant dna coding for a novel protein having beta-1,3-glucanase activity of soya
WO1992017591A1 (en) * 1991-04-08 1992-10-15 Sandoz Ltd. A plant chitinase gene and use thereof
WO1994008009A1 (en) * 1992-10-05 1994-04-14 Mogen International N.V. Antifungal chitin binding proteins and dna coding therefor

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOPRACTICE, Bd.1, 1992 Seiten 33 - 40 JACH, G., ET AL. 'Expression of a bacterial chitinase leads to improved resistance of transgenic tobacco plants against fungal infection' *
BIOTECHNOLOGY, Bd.10, Nr.3, M{rz 1992, NEW YORK US Seiten 305 - 308 LOGEMANN, J., ET AL. 'Expression of a barley ribosome-inactivating protein leads to increased fungal protection in transgenic tobacco plants' *
CURRENT PLANT SCIENCE AND BIOTECHNOLOGY IN AGRICULTURE, VOL.14. ADVANCES IN MOLECULAR GENETICS OF PLANT-MICROBE INTERACTIONS, VOL. 2. 6TH SYMPOSIUM ON MOLECULAR PLANT-MICROBE INTERACTIONS JULY, 1992. Seiten 567 - 571 DUNSMUIR, P., ET AL. 'Resistance to Rhizoctonia-solani in transgenic tobacco' *
DEV. PLANT PATHOL. 2(MECHANISMS OF PLANT DEFENSE RESPONSES), SYMP. HELD AUG. 24-27, 1992 Seite 449 BOJSEN, K., ET AL. 'Genetic transformation of Nicotiana benthamiana with chitinase and beta-1,3-glucanase genes from Beta vulgaris (sugar beet)' *
JOURNAL OF BIOLOGICAL CHEMISTRY, Bd.266, Nr.3, 25. Januar 1991, BALTIMORE, MD US Seiten 1564 - 1573 LEAH, R., ET AL. 'Biochemical and molecular charcterization of three barley seed proteins and antifungal properties' *
SCIENCE, Bd.254, 1991 Seiten 1194 - 1197 BROGLIE, K., ET AL. 'Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani' *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5559034A (en) * 1987-10-02 1996-09-24 Ciba-Geigy Corporation Synergistic antifungal protein and compositions containing same
US5981844A (en) * 1987-10-02 1999-11-09 Novartis Finance Corporation Synergistic antifungal protein and compositions containing same
WO1995018859A1 (en) * 1994-01-10 1995-07-13 Ciba-Geigy Ag Synergistic antifungal protein and compositions containing same
US7087420B1 (en) 1997-07-17 2006-08-08 Cambia Microbial β-glucuronidase genes, gene products and uses thereof
WO2000001846A2 (en) * 1998-07-03 2000-01-13 Devgen N.V. Characterisation of gene function using double stranded rna inhibition
GB2349885A (en) * 1998-07-03 2000-11-15 Devgen Nv Characterisation of gene function using double stranded RNA inhibition
EP1197567A3 (en) * 1998-07-03 2003-01-02 Devgen NV Characterisation of gene function using double stranded RNA inhibition
GB2349885B (en) * 1998-07-03 2003-01-29 Devgen Nv Characterisation of gene function using double stranded RNA inhibition
EP1484415A2 (en) * 1998-07-03 2004-12-08 Devgen NV Method of alleviating pest infestation in plants
WO2000001846A3 (en) * 1998-07-03 2000-06-15 Devgen Nv Characterisation of gene function using double stranded rna inhibition
EP1484415A3 (en) * 1998-07-03 2008-02-13 Devgen NV Method of alleviating pest infestation in plants
EP2045336A3 (en) * 1998-07-03 2009-06-10 Devgen NV Characterisation of gene function using double stranded RNA inhibition
EP2374901A1 (en) * 1998-07-03 2011-10-12 Devgen NV Characterisation of gene function using double stranded RNA inhibition
US8114980B2 (en) 1998-07-03 2012-02-14 Devgen Nv Characterisation of gene function using double stranded RNA inhibition

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HU9302851D0 (en) 1994-01-28
HUT68236A (en) 1995-06-28
US6271438B1 (en) 2001-08-07
DE4234131A1 (en) 1994-04-21
AU4872093A (en) 1994-04-28
ES2201053T3 (en) 2004-03-16
US5804184A (en) 1998-09-08
US20010020300A1 (en) 2001-09-06
MX9306300A (en) 1994-04-29
AU671669B2 (en) 1996-09-05
DE4234131C2 (en) 1995-08-24
DE59310345D1 (en) 2003-07-24
US5689045A (en) 1997-11-18
ZA937202B (en) 1994-04-20
EP0616035B1 (en) 2003-06-18
JPH06197651A (en) 1994-07-19
ATE243258T1 (en) 2003-07-15
HU219268B (en) 2001-03-28
CA2108112A1 (en) 1994-04-10
EP0616035A3 (en) 1995-02-15

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